Hi,

We've done this a lot (granulocytic differentiation), and published with it a lot as well.

Here's my advice:

1. Forget HL60s, as they are fussy and do not do not consistently differentiate into pmn or other types. We found that PLB-985 cells work every time. Neutrophil-like PLB-985 cells have also been better characterized (though perhaps somewhat less used). Fun factoid: there is some evidence that the PLB-985s may actually be a subset from HL-60s. Whatever the case, PLBs work much more reliably, at least if you use cells of know passages (see my next point).

2. Always get your cells from the source (ATCC for the HL-60xs or DSMZ for the PLB-985s). Using cells that "someone got from someone" (as we too often see, though this may not be your case) is bound to land you in trouble. Buying your cells initially is also the only way to make sure you work within the optimal number of passages. PLB-985s can be used up until p20, no more. 

3. Your culture medium (RPMI 1640) and FCS (10% final) must be endotoxin-free. I cannot stress this enough, if you want to have cells that are not pre-activated. 

4. Include 1.25% DMSO in the medium for differentiation, and change the medium every second day (important). The DMSO itself must be for cell culture, not from some large flask. The right stuff comes in glass ampullae under argon. Keep it under an inert gas atmosphere once an ampulla has been opened (you can flush it with nitrogen before re-seailing). Otherwise your DMSO will oxidize within days and its addition to cells will cause small calcium fluxes. You know what that means, so you want to avoid that.

5. Differentiate PLB-985s (w DMSO) for 6 days. You need no particular markers, as most cells will feature the typical polylobed nuclei of neutrophils by d6. If you still want to check for markers, CD11b is always there, so it's a bad choice. You can look for TLR4 or FPR (by FACS), or test responsiveness to LPS or fMLP – uundiff cells do not respond, whereas d6 cells do so very well. 

That should do it.

Viel Glück! 

Hi Sudip,

Some members of my lab previously used differentiated HL60 cells to study neutrophil response under infection. Both, 0.5M ATRA alone as well combining it with 1% DMSO worked fine for us. It's better to check for markers at different intervals during the differentiation process to know after how many days you can get "maximum live and differentiated cells". Only then you'll be able to know what kind of treatment works best in your hands.

I'm not sure what you mean when you say you want to differentiate them "in suspension" since HL-60 cells remain in the suspension and do not adhere. Therefore, differentiation can begin inside the culture flask as it is. Finally, as for the markers, combination of CD11b and CD66b is recommended since any one marker might not be accurate. 

Also, you might want to sort out the dHL-60 population before study chemotaxis and Ca2+ flux since only a limited number of cells would transform and majority cells continue to remain undifferentiated.

Hope it helps! Good luck.

As I know, they do not express CD66b ever, and CD11b is also expresaed before being differentiated. 

Thank you Riva, for the detailed answer. I was unaware of the fact that even after differentiation they do not adhere. 

I have worked extensively with human primary PMNs, which once sticks to any TC surface and activated, they are very hard to detach. Nevertheless, good to know that I do not have to care about this issue anymore. 

About the rest, I will try what you suggested. I did a control experiment before differentiation and found no expression of the markers. I have started a differentiation process with 1% DMSO and will look for CD66b/CD11b markers.

Hi Sudip,

I attempted working with HL-60's, but had a low population that consistently differentiated using 1-1.5% DMSO. I switched to NB4 cells to look at chemotaxis, ROS, F-Actin polymerization, etc.  I use 1uM ATRA (added directly to 2.5x10^5 cells in culture) to differentiate the cells to a PMN-like profile.  The suspension is used on day 5 and 6 for experiments. I have found NB4's very consistent and easy to work with. 

I have a bench flow cytometer, and I check CD11b, CD71, and CD35 for differentiation.  Because I use flow for our Migration Assay, it is easy to determine which cells are alive / dead or differentiated / undifferentiated.  Invitrogen sells a flow kit for live/dead population sorting if you have access to a flow machine.

Good Luck!

Thank you Laura, I will consider your suggestion.

Hello Zhichao,

That is worrisome to know. I would expect a granulocyte-like cell to express CD66b and CD11b both, especially after activation.

Hi,

We've done this a lot (granulocytic differentiation), and published with it a lot as well.

Here's my advice:

1. Forget HL60s, as they are fussy and do not do not consistently differentiate into pmn or other types. We found that PLB-985 cells work every time. Neutrophil-like PLB-985 cells have also been better characterized (though perhaps somewhat less used). Fun factoid: there is some evidence that the PLB-985s may actually be a subset from HL-60s. Whatever the case, PLBs work much more reliably, at least if you use cells of know passages (see my next point).

2. Always get your cells from the source (ATCC for the HL-60xs or DSMZ for the PLB-985s). Using cells that "someone got from someone" (as we too often see, though this may not be your case) is bound to land you in trouble. Buying your cells initially is also the only way to make sure you work within the optimal number of passages. PLB-985s can be used up until p20, no more. 

3. Your culture medium (RPMI 1640) and FCS (10% final) must be endotoxin-free. I cannot stress this enough, if you want to have cells that are not pre-activated. 

4. Include 1.25% DMSO in the medium for differentiation, and change the medium every second day (important). The DMSO itself must be for cell culture, not from some large flask. The right stuff comes in glass ampullae under argon. Keep it under an inert gas atmosphere once an ampulla has been opened (you can flush it with nitrogen before re-seailing). Otherwise your DMSO will oxidize within days and its addition to cells will cause small calcium fluxes. You know what that means, so you want to avoid that.

5. Differentiate PLB-985s (w DMSO) for 6 days. You need no particular markers, as most cells will feature the typical polylobed nuclei of neutrophils by d6. If you still want to check for markers, CD11b is always there, so it's a bad choice. You can look for TLR4 or FPR (by FACS), or test responsiveness to LPS or fMLP – uundiff cells do not respond, whereas d6 cells do so very well. 

That should do it.

Viel Glück! 

Just to add to the confusion... PLB-985 cells are identical to HL-60 cells as determined by DNA fingerprinting (see https://www.dsmz.de/catalogues/details/culture/ACC-139.html and http://igrcid.ibms.sinica.edu.tw/cgi-bin/cell_line_view.cgi?cl_name=PLB985) but maybe they seem to differentiate better. And we look for CD14 as a marker of differentation. 

Not sure how much this helps your experiment, but the following is good to know when working with HL-60.

HL-60 cells (differentiated or undifferentiated) can be made to adhere to tissue culture surfaces by plating them in serum-free media for about 10 minutes. Reference: http://www.sciencedirect.com/science/article/pii/S000326971400325X 

This is not a strong adhesion. Number of adherent cells will greatly reduce even with traces of serum, and they will detatch again in some time. Using poly-L-Lysine coated plates/coverslips increases strength of adhesion, but not by much.

Patrick,

Thank you for the comprehensive answer. I will try my best.

Best,

Sudip